Nongenomic estrogen effects on nitric oxide synthase activity in rat adipocytes.

Estrogens exert multiple genomic effects on adipose tissue through binding to nuclear estrogen receptors. However, there is evidence for additional nongenomic mechanisms whereby estrogens may exert their control on adipose tissue metabolism through rapid activation of various membrane-initiated kinase cascades. Here, we tested rapid effects of estrogens on nitric oxide production in white adipose tissue using 17-beta estradiol (E2) and its membrane impermeant albumin conjugated form (17-beta estradiol hemisuccinate BSA, E2-BSA). We found that both E2 and E2-BSA stimulate nitric oxide synthase (NOS) activity in adipocytes. These effects were abolished by 1) ICI 182-780, a selective estrogen receptor antagonist; 2) wortmannin, an inhibitor of phosphatidylinositol 3-kinase; and 3) N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89) an inhibitor of protein kinase A. In contrast to NOS activation by E2, E2-BSA-induced NOS activity was abolished by UO126, an inhibitor of MAPK kinase/ERK (p42/p44 MAPKs). Immunoblotting studies have shown that both estrogens phosphorylate endothelial NOS (NOS III) on Ser(1179), an effect that is prevented by wortmannin and H89, suggesting that NOS III is the target for estrogen-induced NOS activity. Furthermore, only the E2-BSA-induced NOS III phosphorylation on Ser(1179) was totally abolished by UO126. These results indicate that the signaling cascades involved in adipocyte NOS stimulation by estrogens are different depending on whether estrogens are free or conjugated to albumin and therefore underline the importance of estrogen receptor locations in the nongenomic actions of estrogens in these cells.

Leptin-induced nitric oxide production in white adipocytes is mediated through PKA and MAP kinase activation.

Leptin injection increases plasma levels of nitrites and/or nitrates, an index of nitric oxide (NO) production. Because plasma levels of NO are correlated with fat mass and because adipose tissue is the main source of leptin, it seems that adipose tissue plays a major role in NO release induced by leptin. Adipocytes express both leptin receptors and nitric oxide synthase (NOS; including the endothelial isoform, NOS III, and the inducible isoform, NOS II). In this study, we have demonstrated that physiological concentrations of leptin stimulate NOS activity in adipocytes. This effect of leptin is abolished by 1) AG490, an inhibitor of Janus tyrosine kinase 2/signal transducer and activator of transcription 3; 2) U0126, an inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (p42/p44 MAPK); and 3) N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) or Rp diastereomer of adenosine 3',5'-cyclic phosphorothioate, two inhibitors of protein kinase A, but not by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Immunoblotting studies have shown that leptin fails to activate Akt but increases p42/p44 MAPK phosphorylation, an effect that is prevented by U0126 but not by H-89. Furthermore, leptin induces NOS III phosphorylation at Ser(1179) and Thr(497), but not when adipocytes are pretreated with H-89 or U0126. Finally, stimulation of adipocyte NOS activity by leptin is either unaltered when protein phosphatase 2A is inhibited by 1 nM okadaic acid or completely abolished when protein phosphatase 1 (PP1) activity is inhibited by 3 nM tautomycin, which supports a crucial role for PP1 in mediating this effect of leptin. On the whole, these experiments demonstrate that NOS activity is a novel target for leptin in adipocytes and that the leptin-induced NOS activity is at least in part the result of NOS III phosphorylations via both protein kinase A and p42/p44 MAPK activation. More generally, this study also leads to the hypothesis of NO as a potentially important factor for leptin signaling in adipocytes.

Insulin stimulates nitric oxide production in rat adipocytes.

In adipocytes, insulin regulates the activity of different protein kinases (PI3K/Akt, MAPK, PKC) and protein phosphatases (PP-1, PP-2A). Since these enzymes are implicated in the regulation of NOS activity which is present in adipose tissue, we tested the effects of insulin on white adipocyte NOS activity. Exposure of adipocytes to insulin resulted simultaneously in NOS activity stimulation and Akt activation with maximal effect observed at 1 nM. Higher concentrations of insulin induced a progressive decline of NOS activity. In the presence of wortmannin, a PI3K inhibitor, 1 nM insulin failed to stimulate NOS activity. Insulin (1 nM)-stimulated NOS activity was also abolished by U0126, an inhibitor of p42/p44 MAPK activation, and by 1 microM okadaic acid (OA), which inhibits both PP-1 and PP-2A but not by 1 nM OA which inhibits only PP-2A. Moreover, inhibition of cPKC allowed a high (1 microM) insulin concentration to stimulate NOS activity. These results (i) demonstrate that insulin activates NO production in adipocytes through both PI3K/Akt and MAPK/PP-1 activation and (ii) suggest that PP-1 activation protects NOS against the inhibitory effect of cPKC activation.